Nonspecific recognition is achieved in Pot1pC through the use of multiple binding modes

Abstract

Pot1 is the protein responsible for binding to and protecting the 3' single-stranded DNA (ssDNA) overhang at most eukaryotic telomeres. Here, we present the crystal structure of one of the two oligonucleotide/oligosaccharide-binding folds (Pot1pC) that make up the ssDNA-binding domain in S. pombe Pot1. Comparison with the homologous human domain reveals unexpected structural divergence in the mode of ligand binding that explains the differing ligand requirements between species. Despite the presence of apparently base-specific hydrogen bonds, Pot1pC is able to bind a wide range of ssDNA sequences with thermodynamic equivalence. To address how Pot1pC binds ssDNA with little to no specificity, multiple structures of Pot1pC bound to noncognate ssDNA ligands were solved. These structures reveal that this promiscuity is implemented through new binding modes that thermodynamically compensate for base-substitutions through alternate stacking interactions and new H-bonding networks.

Figure 1

Pot1pC+9mer structure overview. (A) Crystal structure of Pot1pC bound to its cognate 9mer substrate d(GGTTACGGT). 9mer is depicted as lavender sticks and its electron density is contoured to 1.5σ. Pot1pC is colored N to C-terminus (blue to red). Pot1pC is an OB-fold, and 9mer lies across the canonical ligand-binding surface augmented by L23 and L56. (B) Surface representation of Pot1pC in which the DNA and surface atoms within 5 A are colored by element (C-green/violet, O-red, N-blue, P-orange, Se-yellow). This depiction highlights the chemical diversity of the binding interface that includes hydrophobic and polar contacts. Additionally, this depiction illustrates the wide binding pocket and exposed surface between L12 and nucleotides 2–4. Figures were created with MacPyMOL(Schrodinger, LLC, 2010). See also Supplemental Figure S1.

Figure 2

The Pot1pC+9mer interface consists of extensive H-bonding and stacking interactions. (A) The G1 binding pocket involves a stacking interaction with Trp72 and four direct intermolecular H-bonds. (B) Bases 2, 3, and 4 form an off-centered stack. G2 forms three direct H-bonds while T3 forms one direct and one water-mediated H-bond. (C) Leu101 forms the top of the T4 binding pocket while T4 forms two intermolecular and one intramolecular H-bond. A5 stacks between Arg68 and Phe47 and forms three water mediated H-bonds with Pot1pC. (D) An ~90° kink in the DNA orients the backbone towards L23 while bases 6 and 7 point towards the solvent filled portion of the binding pocket. Despite this orientation, C6 still forms two base-specific water-mediated intramolecular H-bonds. (E) Bases G7 and T9 stack between Trp27 and Tyr28. The Watson-Crick face of G7 is solvent exposed, but one direct and one water-mediated H-bond are formed along the Hoogsteen face. T9 forms two direct and one water-mediated H-bond with the protein backbone. (F) G8 is flipped out of the main binding pocket, but is packed between G7 and Arg57. Additionally, G8 forms an extensive array of both inter- and intramolecular H-bonds. See also Supplemental Figure S3.

Figure 3

Alignment of hOB2(Lei et al., 2004) (yellow) and Pot1pC (green) highlight the unexpected ssDNA-binding mode. (A) The beta-barrel cores of the proteins align well, but L23 adopts an extended conformation in Pot1pC (purple) while L23 in hOB2 (red) lies in the canonical ssDNA-binding pocket. (B) The positioning of L23 in the human structure obscures the surface along which the majority of the DNA lies in the S. pombe structure. This causes the human DNA to bind along a unique binding surface with the position of only one base conserved between species.

Figure 4

Complementary base substitution can be accommodated by local adjustments that break and reform H-bonds, but maintain the global binding interface and retain high affinity. (A) The phosphate backbone of T3A (yellow) is altered slightly compared to the original 9mer substrate (lavender), but the protein backbone is unaffected. (B) The adenine substitution at position 3 shifts the base such that two H-bonds are broken and one new H-bond is formed. (C) The A5T complex (orange and white) is globally similar to the cognate (green and lavender). (D) The substituted thymine stacks between Arg68 and Phe47, but two H-bonds are lost relative to the cognate binding mode. (E) The C6G complex (yellow and blue) is globally similar to the cognate (green and lavender). (F) The guanine at position 6 is in the same plane, but rotated 90 degrees relative to the cytosine. This orients the Watson-Crick face towards the protein, which forms a new H-bond with Tyr136. Another new intramolecular H-bond is formed with its own phosphate group while the intramolecular H-bond is maintained with T3. (G) The G8C substrate (blue) is accommodated by a local shift in L23 of Pot1pC (pink) relative to the cognate complex (green and lavender). (H) The cytosine at position 8 is rotated 90 degrees relative to the guanine in the original binding mode. This maintains the hydrophobic and stacking interactions, but creates a new network of hydrogen bonds that is unique from the original substrate (lavender). See also Supplemental Figure S2.

Figure 5

Pot1pC binds 9mer T4A in an alternate binding mode. (A) Alignment of Pot1pC+9mer (green and lavender) and pot1pC+9mer T4A (blue and gray) shows that L23 adopts a new conformation in the T4A binding mode, but the majority of the protein remains unchanged. The DNA maintains the same general contact surface, but the positioning of the majority of bases is altered to some extent. (B) The adenine base at position 4 flips down into the previously unfilled portion of the binding pocket. This new conformation stacks with Arg68, but results in steric clashes with bases T3 and C6 (illustrated by spheres representing the Van der Waals radii of relevant atoms). (C) The steric clash with T3 causes the base to shift and disrupt two H-bonds. This is compensated by an improved stacking interaction between G2 and His100. (D) The steric clash with C6 causes a shift that disrupts the network of H-bonds between the phosphate backbone and L23. L23, however, is able to adopt a new conformation that forms an equally extensive network of Hbonds. (E) The shifts at C6 and L23 disrupt the H-bond network involving G8, but the base is able to rotate over 90° to form a new network of H-bonds. This new network involves amino acids Thr26 and Glu85, which were previously not involved in binding. (F) The shift at position 6 propagates down to the stack involving G7, Trp27, T9, and Tyr28. These bases and amino acids, however, are able to rotate slightly to maintain the majority of stacking and H-bonding interactions seen previously.